Tissue culturing is used in the propagation of new plant varieties, the production of doubled haploids, cryopreservation, conservation of rare and endangered plants, cultivation of difficult-to-propagate plants, and the production of secondary metabolites and transgenic plants. Tissue culturing focuses on the production of high quality, disease-free plant materials for the growth of crop plants and fruit trees. However, major challenges are still associated with the production and distribution of high quality plant materials for plant breeding and the rapid production of improved plants. Currently, tissue culturing is being used particularly for large-scale plant multiplication and micro-propagation, techniques which have wide applications in forestry and agriculture. Hundreds of commercial micro-propagation laboratories worldwide are currently multiplying large number of clones of desired varieties and local flora (IAEA, 2004).
Different opinions among members of the general public have been established regarding plant transformation, particularly by those highly concerned about environmental issues. By contrast, scientists recognize the plant that results from a specific targeted genetic alteration is indistinguishable from the plant that has been developed by a process of breeding and selection. The only difference is that the process of altering a target plant can be greatly accelerated because the genetic modifications can be directed rather than random. Directed modification by homologous recombination has been tested with homologous-recombination dependent gene targeting (hrdGT) (Doetschman et al., 1987) (Gupta & Dhugga, 2010). The problem with this approach is that the relative rate of homologous recombination compared to the rate of random insertion by illegitimate recombination is lower in plant cells than it is in animal cells. Efforts to address this limitation by the expression of foreign genes in plant cells have been made. These methods have had limited success in producing effective gene targeting. Moreover, even when these modified cells are used to effect homologous recombination, the resultant modified cell would still contain an exogenous gene used to select the homologous recombinants and thus still be considered a genetically modified plant by regulators and environmentally concerned entities.
Other methods in which homologous recombination is not involved and the utilization of specific recombination sites and recombinases derived from transposons have also been described in WO 01/85969 and WO 99/25821. The problem with this approach is the mixed structure of the oligonucleotide would likely prevent true recombination by genomic integration.
One of the most common techniques to genetically hybridize plants is the use of plasmid-carrying Agrobacterium tumefaciens. A part of the life cycle of the A. tumefaciens plasmid involves infection of plants. A. tumefaciens introduces the plasmid into the nuclei of plant cells in the form of single strands. A recombinant A. tumefaciens plasmid can be used to introduce exogenous DNA into a plant cell.
Different bacterial plasmid gene treatments have also been used. For example, a simple DNA recombinant plasmid or plasmid holding specific gene cassettes to ensure homologous recombination has been used. Plants hybridized by this method would be classified as genetically-modified organisms (GMOs) and would still be a problem for the food and pharmaceutical industries.
Methods for increasing the transgenic plant size and yield as well as delaying flowering in the plants, using nucleic acids that encode plant transcription factors, have been established (U.S. Pat. No. 7,858,848). However, these methods also involve genetically transforming the plants. Moreover, the extraction of secondary metabolites usually requires high initial amounts of plant biomass or material. In general, the extraction of plant metabolites is carried out from large amounts of fresh biomass material, which requires agronomic practices, the use of chemicals, and time consuming and expensive extraction methods.